Enzymatic synthesis of sulfated polysaccharides without iduronic acid residues

Abstract

Iduronic acid (IdoUA)-free heparan sulfate (HS)-like compounds are provided. Also provided are methods of synthesizing IdoUA-free HS-like compounds. The methods can include providing at least one O-sulfotransferase (OST) enzyme and a reaction mixture comprising 3′-phosphoadenosine 5′-phosphosulfate (PAPS); and incubating a polysaccharide substrate with the at least one OST and the reaction mixture, whereby the HS-like compounds are synthesized. Also disclosed are methods of synthesizing a library of HS-like compounds and methods of determining the mechanism of activity of HS-like compounds.

1 . An iduronic acid (IdoUA)-free heparan sulfate (HS)-like compound comprising the following structure: 
       
         
           
           
               
               
           
         
       
       wherein X is NH or O, R 1  is H, SO 3 H or CH 3 , R 2  is SO 3 H, R 3  is H or SO 3 H, R 4  is H, SO 3 H, CH 3  or CH 2 CH 3 , R 5  is H, SO 3 H, CH 3  or CH 2 CH 3 , R 6  is COOH, COOCH 3 , COOC 2 H 5 , CH 2 OSO 3 H, or CH 2 SO 3 H, L 1  is an alpha or beta linkage, L 2  is an alpha or beta linkage, Y is O or N, and n is ≧4. 
     
     
         2 . The IdoUA-free HS-like compound of  claim 1 , wherein X is NH, R 1  is SO 3 H, R 2  is SO 3 H, R 3  is SO 3 H, R 4  is H or SO 3 H, R 5  is H, L 1  is an alpha or a beta linkage, L 2  is an alpha or a beta linkage, and n is ≧4. 
     
     
         3 . The IdoUA-free HS-like compound of  claim 2 , wherein L 1  is an alpha linkage and L 2  is a beta linkage. 
     
     
         4 . The IdoUA-free HS-like compound of  claim 2 , having a binding affinity to antithrombin ranging from about 20 to about 60 nM. 
     
     
         5 . The IdoUA-free HS-like compound of  claim 2 , having an anti-Xa activity ranging from about 10 to about 500 ng/ml. 
     
     
         6 . The IdoUA-free HS-like compound of  claim 2 , having an anti-IIa activity ranging from about 5 to about 200 ng/ml. 
     
     
         7 . The IdoUA-free HS-like compound of  claim 2 , wherein the HS-like compound has substantially reduced cell proliferation activity as compared to a HS-like compound with iduronic acid residues. 
     
     
         8 . The IdoUA-free HS-like compound of  claim 1 , wherein R 4  is selected from the group consisting of CH 3 , CH 2 CH 3  and SO 3 H and R 5  is selected from the group consisting of CH 3 , CH 2 CH 3  and SO 3 H. 
     
     
         9 . The IdoUA-free HS-like compound of  claim 1 , wherein X is O and R 1  is SO 3 H. 
     
     
         10 . The IdoUA-free HS-like compound of  claim 1 , wherein L 1  is an alpha linkage and L 2  is an alpha linkage. 
     
     
         11 . The IdoUA-free HS-like compound of  claim 1 , wherein L 1  is a beta linkage and L 2  is a beta linkage. 
     
     
         12 . A method of synthesizing an IdoUA-free HS-like compound, comprising incubating a polysaccharide substrate with a sulfo donor compound and an enzyme mixture, the enzyme mixture comprising O-sulfotransferase (OST) enzymes 6-OST-1,6-OST-3 and 3-OST-1 and substantially no epimerase enzyme, wherein synthesis of the IdoUA-free HS-like compound from the polysaccharide substrate is accomplished. 
     
     
         13 . The method of  claim 12 , wherein the polysaccharide substrate is selected from the group consisting of N-sulfo heparosan and chemically desulfated N-sulfated heparin. 
     
     
         14 . The method of  claim 12 , wherein the OST enzymes are recombinant OST enzymes. 
     
     
         15 . The method of  claim 14 , wherein the recombinant OST enzymes are produced in a bacterial expression system. 
     
     
         16 . The method of  claim 12 , wherein the sulfo donor compound comprises a compound capable of donating a sulfonate or sulfuryl group. 
     
     
         17 . The method of  claim 12 , wherein the polysaccharide substrate is partially sulfated prior to incubation. 
     
     
         18 . The method of  claim 12 , further comprising:
 (a) providing a reaction mixture comprising adenosine 3′,5′-diphosphate (PAP), a PAPS regenerating enzyme and a sulfo donor compound; and   (b) incubating the reaction mixture for a time period sufficient to catalyze the production of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) from the PAP by the PAPS regenerating enzyme utilizing the sulfo donor compound as a sulfo substrate,   whereby the sulfo donor compound is provided.   
     
     
         19 . The method of  claim 18 , wherein the PAPS regenerating enzyme is an arylsulfotransferase. 
     
     
         20 . The method of  claim 19 , wherein the arylsulfotransferase is AST-IV. 
     
     
         21 . The method of  claim 18 , wherein the PAPS regenerating enzyme is an estrogen sulfotransferase. 
     
     
         22 . The method of  claim 18 , wherein the sulfo donor compound is an aryl sulfate compound. 
     
     
         23 . The method of  claim 22 , wherein the aryl sulfate compound is p-nitrophenol sulfate (PNPS). 
     
     
         24 . The method of  claim 18 , wherein the time period is from about 1 minute to about 30 minutes. 
     
     
         25 . The method of  claim 18 , wherein the OST enzymes further comprise 2-OST. 
     
     
         26 . The method of  claim 25 , wherein the 2-OST enzyme is a recombinant OST enzyme. 
     
     
         27 . The method of  claim 26 , wherein the recombinant OST enzyme is produced in a bacterial expression system. 
     
     
         28 . A method of synthesizing a library of HS-like compounds, the method comprising incubating polysaccharide substrates with a sulfo donor compound and one or more combinations of biosynthetic enzymes, the biosynthetic enzymes comprising epimerases, N-sulfotransferases and O-sulfotransferases. 
     
     
         29 . The method of  claim 28 , wherein the polysaccharide substrate is N-sulfo heparosan. 
     
     
         30 . The method of  claim 28 , wherein the O-sulfotransferases are selected from the group consisting of 2-OSTs, 3-OSTs and 6-OSTs. 
     
     
         31 . The method of  claim 28 , wherein the O-sulfotransferase enzymes are recombinant OST enzymes. 
     
     
         32 . The method of  claim 31 , wherein the recombinant OST enzymes are produced in a bacterial expression system. 
     
     
         33 . The method of  claim 28 , wherein the polysaccharide substrate is partially sulfated prior to incubation. 
     
     
         34 . The method of  claim 28 , further comprising:
 (c) providing a reaction mixture comprising adenosine 3′,5′-diphosphate (PAP), a PAPS regenerating enzyme and a sulfo donor compound; and   (d) incubating the reaction mixture for a time period sufficient to catalyze the production of 3′-phosphoadenosine 5′-phosphosulfate (PAPS),   whereby the sulfo donor compound is provided.   
     
     
         35 . The method of  claim 34 , wherein the PAPS regenerating enzyme is an arylsulfotransferase. 
     
     
         36 . The method of  claim 35 , wherein the arylsulfotransferase is AST-IV. 
     
     
         37 . The method of  claim 34 , wherein the PAPS regenerating enzyme is an estrogen sulfotransferase. 
     
     
         38 . The method of  claim 34 , wherein the sulfo donor compound is an aryl sulfate compound. 
     
     
         39 . The method of  claim 38 , wherein the aryl sulfate compound is p-nitrophenol sulfate (PNPS). 
     
     
         40 . The method of  claim 34 , wherein the time period is from about 1 minute to about 30 minutes. 
     
     
         41 . The method of  claim 34 , wherein the polysaccharide substrate is a chemically desulfated N-sulfated heparin. 
     
     
         42 . A method of determining a mechanism of activity of HS-like compounds, the method comprising:
 (e) synthesizing one or more HS-like compounds by incubating one or more polysaccharide substrates with a sulfo donor compound and one or more combinations of biosynthetic enzymes, the biosynthetic enzymes comprising epimerases, N-sulfotransferases and O-sulfotransferases;   (f) subjecting one or more of the HS-like compounds to a test for an activity; and   (g) determining a mechanism of activity of the HS-like compounds based on one or more results of the one or more HS-like compounds.   
     
     
         43 . The method of  claim 42 , wherein the polysaccharide substrate is selected from the group consisting of N-sulfo heparosan and chemically desulfated N-sulfated heparin. 
     
     
         44 . The method of  claim 42 , wherein the O-sulfotransferases are selected from the group consisting of 2-OSTs, 3-OSTs and 6-OSTs. 
     
     
         45 . The method of  claim 42 , wherein the O-sulfotransferase enzymes are recombinant OST enzymes. 
     
     
         46 . The method of  claim 45 , wherein the recombinant OST enzymes are produced in a bacterial expression system. 
     
     
         47 . The method of  claim 42 , wherein the polysaccharide substrate is partially sulfated prior to incubation. 
     
     
         48 . The method of  claim 42 , wherein the test is for an activity selected from the group consisting of anti-thrombin binding activity, anti-Xa activity, anti-IIa activity, cell proliferation stimulation activity, cell growth stimulation activity, activated partial thromboplastin time, prothrombin time and combinations thereof. 
     
     
         49 . The method of  claim 42 , further comprising:
 (h) providing a reaction mixture comprising adenosine 3′,5′-diphosphate (PAP), a PAPS regenerating enzyme and a sulfo donor compound; and   (i) incubating the reaction mixture for a time period sufficient to catalyze the production of 3′-phosphoadenosine 5′-phosphosulfate (PAPS),   whereby the sulfo donor compound is provided.   
     
     
         50 . The method of  claim 49 , wherein the PAPS regenerating enzyme is an arylsulfotransferase. 
     
     
         51 . The method of  claim 50 , wherein the arylsulfotransferase is AST-IV. 
     
     
         52 . The method of  claim 49 , wherein the PAPS regenerating enzyme is an estrogen sulfotransferase. 
     
     
         53 . The method of  claim 49 , wherein the sulfo donor compound is an aryl sulfate compound. 
     
     
         54 . The method of  claim 53 , wherein the aryl sulfate compound is p-nitrophenol sulfate (PNPS). 
     
     
         55 . The method of  claim 49 , wherein the time period is from about 1 minute to about 30 minutes.